The present invention relates to a novel assay to provide a quick and accurate quantitative determination of analytes such as hormones, proteins and enzymes while utilizing the principle of immunochromatographic method.
Indicator substances such as hormones, proteins and microorganisms that enable expectation and progression of human diseases have relatively complex structure, and thus they are mostly determined by immunoassays using antigen-antibody reaction. Such examinations have been generally carried out in specialized laboratories. However, the need of on-site examination at the point-of-care such as hospitals or emergency rooms and even at home, is growing rapidly (Reference: C. P. Price et al., Principles and Practice of Immunoassay, 1997, page 579-603, Macmillan Reference Ltd., London). To this end, immunoassay system which does not require any expert knowledge and complex procedure, and is simple to use and provides quick responses, has been developed, and such diagnostic performance could be somewhat achieved by an immuno-chromatography method, which employs microporous membrane for immobilizing a binding protein (e.g. antibody) (Reference: M. P. A. Laitinen, 1996, Biosens. Bioelectron. Vol. 11, 1207-1214; S. H. Paek et al., 1999, Anal. Lett., Vol. 32, 335-360). When the analyte-containing sample is absorbed from the bottom end of the membrane strip, the analyte is transported to the layer of immobilized binding protein by the capillary action through membrane pores. A binding reaction between the analyte and the binding protein occurs on the surface of solids, and unbound molecules are subsequently separated by the medium flow. As the transfer of reactants is accelerated by the lateral flow of medium, the above membrane immuno-chromatography method provides a quick analysis of analyte and convenience of one-step detection where the analysis is completed upon sample application alone.
An immuno-strip model system to perform immuno-chromatographic assay comprises the following three membrane strips ((a) of FIG. 1). From the bottom, glass fiber membrane (10) for sample application, nitrocellulose (NC) membrane (20) with immobilized binding protein (21) specific to analyte, and cellulose membrane (30) as absorbent are shown. These membrane strips are partially superimposed and arranged on a plastic film and fixed by double-sided tape. Considering the analytical procedure, a conjugate (11) between binding protein and signal generating substance (e.g. gold colloid particles, enzymes, colored plastic beads) is added on a predetermined site of the glass fiber membrane, and a medium (2) containing the analyte (1) is absorbed from the bottom end of immuno-strip. The analyte is transported to the upper part of the strip by the medium flow that is induced by the capillary action, and binds to the conjugate through immune reaction upon contact with binding protein in the conjugate ((b) of FIG. 1). When the medium carries the binding complex to the binding protein immobilized on the NC membrane, the binding complex is captured by the immobilized binding protein through immune reaction (the site on the antigen molecule recognized by this binding protein is different from the recognized by the binding protein conjugated with signal generator ((c) of FIG. 1). Uncaptured components are transferred by the medium flow for separation to the absorbent cellulose membrane. Such sandwich-type complex formed on a defined site on the membrane (complex where two binding protein molecules are bound to both sides of the analyte molecule at the same time) generates a color signal (25) detectable by the naked eye because it includes the indicator, color generating substance.
Such a qualitative analytical mode of the system may be sufficient for the identification of certain physical symptoms or infections causing diseases, but most substances for clinical investigation demand analyte concentration known in each specimen. If quantitation is desired, the color signal can be converted to optical density by adopting a photometric transducing capability, yet because of difficulty in reproducible conversion and poor detection sensitivity, it is not widely used.
Therefore, it has been required to develop a novel quantitative method to determine analyte concentration more simply and accurately relative to photometry, while directly using the immuno-chromatographic method that is simple to use and provides a quick response.
The present inventors have completed the present invention based on the idea that gold colloids used as signal generating substance in the above immuno-chromatography method are metal particles with conductivity.
Thus, the object of the present invention is to provide a more convenient, accurate and novel quantitative analysis. In other words, the object of the present invention is to provide a novel quantitative as well as qualitative method of electrochemical assay with convenience and sensitivity for various analytes such as hormones, proteins and enzymes, while using the principle of immuno-chromatography.